Transient and stable vector transfection: Pitfalls, off-target effects, artifacts.

Transient and stable vector transfections have played important roles in illustrating the function of specific genes/proteins. The general assumption is that such a platform could effectively link a given gene/protein to gained phenotypes, revealing the mechanism of how a gene works. However, in reality, increased studies have surprisingly noticed some unexpected results. In this review, we demonstrate that an assumption that empty vector-transfected cells preserve the cytogenetic and phenotypic characteristics, and represent the adequate control in transfection experiments is not universally valid. A DNA vector, a transfection reagent, expression of an antibiotic resistance (trans)gene, expression of a reporter (trans)gene, and selection by acute/chronic antibiotic treatment may evoke cellular responses that affect the biochemical processes under investigation. We exemplify a number of studies, which reported obvious genomic, transcriptomic and phenotypic changes of tumor cells after transient/stable transfection of an empty vector. To further address the common mechanisms of these unexpected findings, we will apply the genome theory of somatic evolution to explain stress-mediated system dynamics and the limitations of predicting the system behavior solely based on targeted genes. We conceptualize that the diverse experimental manipulations (e.g., transgene overexpression, gene knock out/down, chemical treatments, acute changes in culture conditions, etc.) may act as a system stress, promoting intensive genome-level alterations (chromosomal instability, CIN), epigenetic and phenotypic alterations, which are beyond the function of manipulated genes. Such analysis calls for more attention on the reduced specificities of gene-focused methodologies.